Use of puroindoline for preparing biscuits

ABSTRACT

The present invention relates to the use of puroindolines as additives in biscuit manufacture. More specifically, the present invention relates to the use of puroindolines added to biscuit flour in order to increase the firmness of a biscuit.

The invention relates to the use of puroindolines as additive in biscuitmanufacture.

Puroindolines are proteins which are present in wheat grain and whichhave the capacity to interact with lipids. Two isoforms ofpuroindolines, respectively called puroindoline a and puroindoline b,have been characterized. They are basic proteins (pI ˜10) of lowmolecular weight (about 13 kDa); they comprise 10 cysteine residues,forming 5 disulfide bridges, and a domain rich in tryptophan residues[BLOCHET et al., in: “Gluten Proteins 1990”; (BUSHUK W. and TKACHUK R.,eds), AACC, St Paul Minn., pp 314-325 (1991); BLOCHET et al., FEBSLett., 329: 336-340, (1993).

Moreover, a recent publication [GIROUX and MORRIS, Proceedings of theNational Academy of Sciences, vol. 95 p. 6262-6266, (1998)] indicatesthat the protein called friabilin, considered as a biochemical markerfor wheat grain hardness [GREENWELL and SCHOFIELD, Cereal Chem., 63,369-380 (1985)], is a heterodimer of puroindolines a and b which isinvolved in controlling the hardness of the endosperm.

Puroindolines possess specific surfactant properties linked to theirhigh affinity for lipids; they have in particular a high foaming powerwhich is increased in the presence of polar lipids [DUBREIL et al., J.Agric. Food Chem., 45: 108-116 (1997); WILDE et al., J. Agric. FoodChem., 41, 1570-1576 (1993)]. It has thus, for example, been shown thatthe addition of puroindoline made it possible to restore the foamingproperties of beer supplemented with stearic acid, phospholipids ortriglycerides [CLARK et al., J. Inst. Brew. 100, 23-25 (1994)], or thoseof egg white supplemented with oil [HUSBAND et al., in: “FoodMacromolecules and Colloids” (DICKINSON E. and LODENT D., eds), RoyalSociety of Chemistry, London, pp. 285-296, (1995)].

Recent work relating to the potential applications of the properties ofpuroindolines in the context of the manufacture of bread shows that theaddition of a small quantity of puroindoline (0.1% relative to theweight of the flour) considerably modifies the rheological properties ofthe dough and the structure of the bread crumb [DUBREIL et al., CerealChem. 75, 2: 222-229, (:998)]. however, the effect of puroindolines onthe volume of the final product after baking varies markedly accordingto the baking quality of the flour used (which is linked for a largepart to its glutenin composition). When puroindolines are added to flourwith good baking quality, a reduction of the order of 10% in breadvolume (in other words an increase in its density) is observed comparedwith bread made without addition of puroindolines; this decrease involume reaches 20% if flour of poor baking quality is used. On the otherhand, if puroindolines are added to flour of average baking quality(resulting from the mixing of the above two), an increase in breadvolume of the order of 20% is observed.

The inventors have undertaken the study of the effect of puroindolineson preparations which are more complex than bread dough, such as thedoughs normally used in pastry and biscuit manufacture.

They have thus observed that the addition of puroindoline during thepreparation of biscuits induced a significant effect on the textureand/or the density of the finished product, and that it was possible toindependently control these two parameters.

The subject of the present invention is the use of puroindoline asadditive which makes it possible to control the texture and/or thedensity of a biscuit.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: FIG. 1 displays the results of trials comparing the density ofhard biscuits made from flour containing endogenous puroindolinessupplemented with defined amounts of puroindolines versus the controlgroup of flour containing endogenous puroindolines supplemented withdefined amounts of ovalbumin. The open square (□) represents thequantity of purified puroindolines added to the flour and the darkenedcircle () represents the quantity of ovalbumin added to the flour.

FIG. 2: FIG. 2 displays the results of trials comparing the density ofhard biscuits made from flour containing no endogenous puroindolinessupplemented with 0.2% of puroindolines () versus the control group ofeither flour containing no endogenous puroindolines supplemented with0.2% of ovalburin () or flour alone (□).

FIG. 3: FIG. 3 displays the results of trials comparing the density ofsoft biscuits made from either a) flour containing no endogenouspuroindolines (□), b) flour containing no endogenous puroindolinessupplemented with puroindolines (0.1% by weight) (), or c) flourcontaining 0.1% of endogenous puroindolines ().

FIG. 4: FIG. 4 represents the variation of the mean force (F_(mean))over time for the soft biscuits obtained from the various flours tested:flour containing 0.1% of endogenous puroindolines (▴); flour withoutendogenous puroindolines (♦); and flour without endogenous puroindolinessupplemented with 0.1% (by weight relative to the weight of the flour)of purified puroindolines (▪).

FIG. 5: FIG. 5 displays the results of trials comparing the density ofpuff biscuits made from either a flour containing no endogenouspuroindolines (□) or flour containing no endogenous puroindolinessupplemented with purified puroindolines (0.1% by weight relative to theweight of the flour) ().

FIG. 6: FIG. 6 represents the variation in the mean force (F_(mean))during storage for the puff biscuits made from either a flour containingno endogenous puroindolines (♦) or flour containing no endogenouspuroindolines supplemented with purified puroindolines (0.1% by weightrelative to the weight of the flour) (▪).

FIG. 7: FIG. 7 displays the results of trials comparing the density ofpuff biscuits containing 3% of rapeseed oil made from either a flourcontaining no endogenous puroindolines (□) or flour containing noendogenous puroindolines supplemented with purified puroindolines (0.1 %by weight relative to the weight of the flour) ().

FIG. 8. FIG. 8 represents the variation in the mean force (F_(mean))during storage for the puff biscuits containing 3% of rapeseed oil madefrom either a flour containing no endogenous puroindolines (♦) or flourcontaining no endogenous puroindolines supplemented with purifiedpuroindolines (0.1% by weight relative to the weight of the flour) (▪).

For the purposes of the present invention, the term “puroindoline” isunderstood to mean not only the wheat puroindolines a and b mentionedabove, but also any protein or any peptide possessing functional andstructural characteristics similar to those of said wheat puroindolines,and in particular any protein consisting of at least one polypeptidechain comprising at least 8 cysteine residues involved in disulfidebridges, and a tryptophan-rich domain, and possessing surfactantproperties similar to those of said wheat puroindoline.

This includes in particular any natural or recombinant proteinconsisting of at least one polypeptide chain having the sequence of oneof the allelic variants or isoforms of wheat puroindolines, or ofhomologous proteins present in other cereals such as oats, barley,triticale and rye, or of at least one polypeptide chain comprising atleast the portions characteristic of said sequence, and which arenecessary for the functional properties of puroindolines, as definedabove.

These puroindolines may be used in the form of monomers as well as inthe form of homomeric or heteromeric assemblies.

For the purposes of the present invention, the term “biscuit” isunderstood to mean any product obtained by baking a mixture, generallyin the form of dough, comprising, in addition to flour obtained fromcereal(s), other ingredients providing proteins, carbohydrates and/orlipids; this may include for example proteins of animal origin such asegg or milk proteins, and the like, proteins of plant origin such assoybean proteins, and the like, sugar, fatty substances of animal orplant origin. The dough is prepared from said ingredients according toany of the conventional techniques used in biscuit manufacture, whichare known per se; it is thus possible, for example, to prepare a doughwhich is laminated, or formed by a rotary press, (so as to obtain hardbiscuit-type products), a leavened dough so as to obtain cake-typeproducts), a puff dough (so as to obtain genoese-type products), afermented dough so as to obtain “cracker” type products, and the like.It is also possible to prepare a biscuit by carrying out all or part ofthe mixing, kneading and baking of the ingredients in a cooker-extruder.

Earlier work in the bakery sector mentioned above has shown that theaddition of puroindolines to flours free of endogenous puroindolinesresulted in an increase in the density, and therefore a decrease in thevolume of the bread [DUBREIL et al., publication cited above (1997)]. Itcould therefore be assumed that the use of these same flours,supplemented with puroindolines, in biscuit manufacture would result insimilar effects.

However, the inventors have observed, surprisingly, that the addition ofpuroindoline in the context of the preparation of biscuits only resultedin an increase in density in the specific case of puff biscuits preparedfrom a dough with a high content (that is to say greater than 7%) offat, and could, in this case, be counterbalanced by the addition ofemulsifiers. In the other cases, a decrease in density is on thecontrary observed; moreover, it is observed that the addition ofpuroindolines results in all cases in an increase n the firmness of softbiscuits and of puff biscuits.

The effects of the use of puroindoline may be modulated according to thewater content of the dough and/or its fat content and/or the quantity ofemulsifier which it contains.

The decrease in density induced by the addition of puroindolines is forexample lower in the case of the products obtained from a doughcontaining only a small amount of water (laminated hard biscuits forexample).

The effect on the decrease in density also becomes less important whenthe content of fatty substances increases; as indicated above, for ahigh content of fatty substances, by contrast, an increase in density isobserved in the case of puff biscuits.

However, in puff biscuits containing fat and puroindolines, the decreasein density is promoted by the addition of emulsifiers such as lecithin,mono- and diglycerides of fatty acids, acetic, lactic, citric ortartaric esters of mono- and diglycerides of fatty acids, mono- anddiacetyl tartaric esters of mono- and diglycerides of fatty acids, mixedacetic and tartaric esters of mono- and diglycerides of fatty acids,sucroesters of fatty acids, polyglyceric esters of fatty acids,1,2-propanediol esters of fatty acids, sodium 2-stearoyllactylate,calcium 2-stearoyl-lactylate. The emulsifiers make it possible tocounterbalance the effect of a high content of fatty substances whichare likely to trap the puroindolines.

In accordance with the invention, said puroindoline may for example beused:

to reduce the density of a puff biscuit obtained from a dough whose fatcontent is less than or equal to 4% of the total weight of the dough;

to reduce the density of a hard or soft, non-puff dough biscuit obtainedfrom a dough whose fat content is between 2% and 30%, preferably between2 and 20%, in the case of a hard biscuit, of the total weight of thedough;

to increase the firmness of a soft or puff dough biscuit; this increasein firmness contributes in particular to the obtaining of a feeling ofmelting in the mouth, which is appreciated by consumers.

Puroindoline induces a decrease in density greater than that observed,under the same conditions, with products normally used for reducing thedensity of biscuits, such as ovalbumin; it can therefore beadvantageously used as a replacement for all or some of these products,or of ingredients containing them, in particular eggs. The effect on thereduction in density will be all the more marked as the reduction in thequantity of eggs causes a decrease in the total content of fattysubstances in the finished product. In addition, since eggs constitute ahighly variable raw material in terms of quality, their replacement withpuroindoline therefore allows, for the same density, betterreproducibility of the finished product.

The addition of puroindoline can also make it possible to improve aflour of poor biscuit-making quality, and to obtain from it biscuits ofsimilar density to those obtained from a flour of good biscuit-makingquality.

For the use of the present invention, puroindoline may be added in anamount of 0.02 to 5%, preferably 0.05 to 2% of the dry weight of theflour used for the preparation of the biscuit, according to the initialpuroindoline content of the flour, and the final content which it isdesired to obtain.

It is possible to use a purified puroindoline preparation (containing asingle isoform or a mixture of isoforms); it is also possible to use acereal fraction (for example gluten or starch) enriched withpuroindoline.

The flours previously used in biscuit manufacture have a puroindolinecontent which does not exceed 0.2% (of the dry weight of the flour). Theinvention also includes the use, for the preparation of biscuits, offlours having a puroindoline content greater than this value, andpreferably of between 0.2 and 5%, advantageously between 0.2 and 2% ofthe dry weight of the flour.

The term “flour” is understood to mean not only wheat flour, but alsoflour from other cereals, such as rye, barley, triticale or oats, or amixture of these flours.

It is also possible to use flours enriched with puroindolines byaddition of purified puroindoline, or of a fraction rich inpuroindoline, as indicated above; it is also possible to use floursobtained from varieties of cereals selected on the criterion of theirrichness in puroindoline, or of transgenic cereals overexpressing atleast one of the isoforms of puroindoline.

The present invention also includes the biscuit doughs, as well as thebiscuits which can be obtained from said flours.

The present invention will be understood more clearly with the aid ofthe additional description which follows, which refers to nonlimitingexamples of the use of puroindoline in the preparation of various typesof biscuit.

EXAMPLE 1 Incorporation of Puroindolines into the Flour

A mixture of the a and b isoforms of puroindoline was extracted andpurified using the method described by BLOCHET et al. (1991, publicationcited above), modified by COMPOINT et al. [Large scale and rapidpurification of plant lipid binding proteins by combining TRITON X114phase partitioning and ion exchange chromatography, Conference on PlantProteins from European crops, Nantes, France (1996)]; the preparationobtained is freeze-dried.

The flours enriched with puroindoline are obtained by mixing thefreeze-dried product, which is designated in the examples below by theterm “purified puroindolines”, with the flour.

For the experiments described in the examples below, the mixtures wereproduced, on the one hand, from flour of the type normally used inbiscuit manufacture, containing about 0.1% to 0.15% of endogenouspuroindolines, and on the other hand from flour not containingendogenous puroindoline. When the quantity of flour used is less than300 g, the mixtures are produced directly in the kneader. When thequantity of flour used is greater than 300 g, the mixture is homogenizedbeforehand by sieving the powders (flour+puroindoline) on an 800 μmsieve.

EXAMPLE 2 Effect of the Puroindolines on a Laminated Hard Biscuit

Incorporation of the Puroindolines into the Flour

a) Flour Containing Endogenous Puroindolines

The freeze-dried puroindolines and flour containing 0.15% of endogenouspuroindolines are mixed in a kneader, in the proportions indicated intable 1 below.

TABLE I Quantity of freeze-dried Flour puroindolines added Trial 0% 290g  0 mg Trial 0.05% 290 g 145 mg Trial 0.1% 290 g 290 mg Trial 0.2% 290g 580 mg

In parallel, control mixtures are produced by replacing thepuroindolines with the same quantity of ovalbumin, which is a proteinconventionally used for increasing the volume (and therefore forreducing the density) of biscuits.

b) Flour Containing no Endogenous Puroindoline

Another series of trials is carried out by adding 0.2% of purifiedpuroindolines, or 0.2% of ovalbumin, to a flour containing no endogenouspuroindoline.

For the two series of trials, the flour enriched with puroindolines orwith ovalbumin is used like a conventional flour in the preparation of alaminated hard biscuit, according to the protocol indicated below.

Formula for the Laminated Hard Biscuit

The following ingredients are mixed in the proportions indicated below:

Flour 100 Icing sugar 30.00 Lard 8.00 Ammonium bicarbonate 1.00 Salt0.63 Sodium bicarbonate 0.50 Sodium pyrophosphate 0.50 Water 26.00

The mixture is kneaded for 8 minutes (kneader temperature: 24° C.,kneader speed: 30 rpm).

After resting for 30 minutes, the dough is laminated and cut into doughpieces with a hollow punch.

The dough pieces are baked in an oven at a temperature of 280° C. for 7min.

Density of the Laminated Hard Biscuits

The mass, length and thickness are determined for each biscuit, and thedensity is calculated.

The results of trials a) are illustrated by FIG. 1, which represents thedensity of the biscuits as a function of the quantity of purifiedpuroindolines (□) or of the quantity of ovalbumin () added to the flour(% by weight relative to the weight of the flour).

No significant difference is observed between the biscuits manufacturedfrom flour containing 0.15% of endogenous puroindoline, and thosemanufactured from this same flour enriched with 0.05% of purifiedpuroindolines. By contrast, a decrease in the density of the biscuits ofthe order of 3% is obtained when the biscuits are manufactured fromflour containing 0.15% of endogenous puroindoline enriched with 0.1% or0.2% of purified puroindolines.

The results of trials b) are illustrated by FIG. 2, which represents thedensity of the biscuits obtained from a flour containing no endogenouspuroindoline, with no additive, or supplemented with 0.2% (by weightrelative to the weight of the flour) of purified puroindolines, or ofovalbumin.

Legend to FIG. 2:

Flour with no additive:

Flour+0.2% puroindoline:

Flour+0.2% ovalbumin:

The results of the two series of trials show hat the addition ofpuroindoline induces a decrease in the density of the biscuits which ismarkedly greater than that induced by ovalbumin.

EXAMPLE 3 Effect of the Puroindolines on a Soft Biscuit

The biscuits are made according to the formula (% by weight) indicatedin table II below:

TABLE II FLOUR 30% SUGAR 30% EGGS 4.5% FAT 10% GLYCERIN 4% WATER 18%WHEY 2% BAKING POWDERS 1.1% SALT 0.4% LECITHIN 0.4%

Three different flours are used: a flour containing no puroindoline(control 0% of puroindoline), the same flour to which 0.1% by weight ofpurified puroindolines relative to the weight of flour is added and aflour containing 0.1% of endogenous puroindolines (control endogenouspuroindoline).

For the production of flour enriched with puroindoline the flour issieved with the puroindolines using an 800 μm sieve. This enriched flouris then sieved again with the rest of the powders immediately beforeuse.

These powders are respectively: baking powders, salt, lecithin, sugar,powdered eggs. They are weighed in the same container and mixed with theflour immediately before use.

Mixing with the liquid ingredients is then carried out and the baking iscarried out for 10 min at 180° C.

The density of the biscuits obtained and the variation of their textureduring storage are measured. The measurements are carried out accordingto the following protocols:

Density:

Slices 2 cm in diameter are cut out in the biscuit with a hollow punchand weighed. The density is determined according to the followingformula:

Density=m/(w*r ² h/10)

m=mass

r=radius

h=height of the slices.

Texture:

The texture is measured with the aid of an INSTRON penetrometer; a roll,cut out in the biscuit to be tested, is compressed at a constant rate.The force-movement of compression curve is characteristic of the samplestudied. The measurement of the mean force necessary to compress theproduct makes it possible to determine its firmness.

The measurement parameters are the following:

modulus of compression: roll 2.5 cm in diameter

distance between the plates: 15 mm

rate of traverse: 40 mm/mm

measurement load at origin HO: 0.1 N

minimum safe load: 100 N

maximum (safe) movement: 13

initial compression 0%

final compression: 50%

peak detection criterion: 0.1 N

max. default value 1: 25%

Results:

Density

The results are illustrated by FIG. 3. Legend to FIG. 3:

Flour containing 0.1% of endogenous puroindoline:

Flour without endogenous puroindoline:

Flour without endogenous puroindoline: +0.1% puroindolines:

The biscuits obtained from flour having no endogenous puroindoline aremore dense than the biscuits manufactured from flour containing 0.1% ofendogenous puroindoline. The addition of 0.1% of purified puroindolinesto flour containing no endogenous puroindoline causes a decrease ofabout 6% in the density of the biscuit; the soft biscuits obtained inthis manner have a density similar to that of the biscuits obtained fromflour containing 0.1% of endogenous puroindoline. Texture The resultsare illustrated by FIG. 4 which represents the variation of the meanforce (Fmean) over time, for the biscuits obtained from the variousflours tested; flour containing 0.1% of endogenous puroindoline (▴);flour without endogenous puroindoline (♦); flour without endogenouspuroindoline supplemented with 0.1% (by weight relative to the weight ofthe flour) of purified puroindolines (∪).

These results show that the addition of puroindoline has a significanteffect on the texture of the biscuit. The biscuits obtained from theflour without endogenous puroindoline supplemented with purifiedpuroindolines have a firmer texture after 29 and 56 days of storage thanthe biscuits obtained from flour containing 0.1% of endogenouspuroindoline, or from the flour without endogenous puroindoline. From 80days of storage (which is more than the period normally separating themanufacture of the product from its consumption), the difference intexture between the products is no longer significant.

EXAMPLE 4 Effect of Puroindolines on a Puff Biscuit

The biscuits are made according to the formula indicated in table IIIbelow:

TABLE III In % In grams Flour 35 500 Granulated sugar 35 500 Liquidwhole eggs 30 450

Two different flours are used: a flour containing no endogenouspuroindoline, and the same flour enriched with 0.1% by weight ofpurified puroindolines relative to the weight of the flour.

The biscuit dough thus obtained contains about 4% of fatty substancesprovided by the eggs and the flour.

The density and the texture of the biscuits obtained are measured asindicated in example 3 above.

Results

Density

The results are illustrated by FIG. 5. Legend to FIG. 5:

Flour without endogenous puroindolines:

Flour without endogenous puroindolines +0.1% puroindolines:

These results show that the addition of 0.1% of purified puroindolinescauses a decrease of 14% in the density of the biscuits.

Texture

The results are illustrated by FIG. 6 which represents the variation inthe mean force (Fmean) during storage, for the biscuits obtained fromflour with no endogenous puroindoline (♦), or from flour with noendogenous puroindoline, supplemented with 0.1% (by weight relative tothe weight of the flour) of purified puroindolines (▪).

The puff biscuits containing purified puroindolines possess a firmertexture, which results in a firmness greater than 13%, at 61 days ofstorage. This effect is greater than that, described in Example 3,observed in the case of soft biscuits.

EXAMPLE 5 Effect of Puroindolines on a Puff Biscuit Containing 3% ofRapeseed Oil

Incorporation of the Puroindolines into the Flour

The biscuits are made according to the formula indicated in table IVbelow:

TABLE IV In % In gram Flour 33.5 500 Rapeseed oil 3  50 Granulated sugar33.5 500 Liquid whole eggs 30 450

Two different flours are used: a flour containing no endogenouspuroindoline, and the same flour enriched with 0.1% of purifiedpuroindolines.

The rapeseed oil is added before the overrun stage.

The biscuit dough thus obtained contains about 7% of fatty substances,making into account those provided by the eggs and the flour.

The density and the temperature of the biscuits obtained are measured asindicated in Example 3 above. Density

The results are illustrated by FIG. 7. Legend to FIG. 7:

Flour without endogenous puroindolines:

Flour without endogenous puroindolines +0.1% puroindolines:

The addition of 0.1% of purified puroindolines to the flour containingno endogenous puroindoline results in a significant increase, of theorder of 20%, in the density of the biscuits.

Texture

The results are illustrated by FIG. 8 which represents the variation inthe mean force (F_(mean)) during storage, for the biscuits obtained fromflour without endogenous puroindoline (♦), or from flour withoutendogenous puroindoline, supplemented with 0.1% (by weight relative tothe weight of the flour) of purified puroindolines (▪).

The biscuits obtained from the flour supplemented with puroindolineshave, at 35 days of storage, a firmness 65% greater than that of thebiscuits obtained from the flour without puroindoline.

The results obtained show that the effect of the puroindolines on thedensity of the puff biscuits depends on the fat content of the formula.It is thus possible to use puroindolines to decrease or on the contraryto increase the density of this type of product, according to its fatcontent.

The effect of the puroindolines on the texture also appears to be linkedto the fat content; however, it is always in the same direction, that isto say toward an increase in firmness.

What is claimed is:
 1. A method of making soft or puff dough biscuitsfrom a dough comprising flour and additional ingredients providingproteins, carbohydrates, and lipids, wherein the improvement comprisesadmixing the dough with at least one puroindoline; and wherein the atleast one puroindoline is added in an amount effective for increasingthe firmness of the biscuits resulting from baking the dough.
 2. Themethod of claim 1 wherein the amount of puroindoline added is effectiveto reduce the density of a soft biscuit prepared from a dough with a fatcontent of between 2 and 30% of the total weight of the dough.
 3. Themethod of claim 1 wherein the amount of puroindoline added is effectiveto reduce the density of a puff biscuit prepared from a dough with a fatcontent less than or equal to 4% of the total weight of the dough. 4.The method of claim 1 wherein the amount of puroindoline added iseffective to increase the density of a puff biscuit prepared from adough without added emulsifier and which dough has a fat content greaterthan or equal to 7% of the total weight of the dough.
 5. A method ofmaking soft or puff dough biscuits from a mixture comprising flour andadditional ingredients providing proteins, carbohydrates, and lipids,wherein the improvement comprises admixing the flour with at least onepuroindoline; and wherein the at least one puroindoline is added in anamount effective for increasing the firmness of the biscuits resultingfrom baking the mixture.
 6. The method of claim 5 wherein the amount ofpuroindoline added is between 0.02 and 5% by weight relative to theweight of the flour.
 7. The method of claim 5 wherein the flour has apuroindoline content greater than 0.2% of the dry weight of the flourwith the additional ingredients.
 8. The method of claim 7 wherein thepuroindoline content of the flour is between 0.2 and 2% of the dryweight of the flour.
 9. A method of making non-puff dough biscuits froma dough comprising flour and additional ingredients providing proteins,carbohydrates, and lipids, wherein the improvement comprises admixingthe dough with at least one puroindoline; and wherein the at least onepuroindoline is added in an amount effective for reducing the density ofthe biscuits resulting from baking the dough.
 10. The method of claim 9wherein the amount of puroindoline added is effective to reduce thedensity of a hard biscuit prepared from a dough with a fat content ofbetween 2 and 20% of the total weight of the dough.
 11. The method ofclaim 9 wherein the amount of puroindoline added is effective to reducethe density of a biscuit prepared from a dough with a fat content ofbetween 2 and 30% of the total weight of the dough.
 12. A method ofmaking non-puff dough biscuits from a mixture comprising flour andadditional ingredients providing proteins, carbohydrates, and lipids,wherein the improvement comprises admixing the flour with at least onepuroindoline; and wherein the at least one puroindoline is added in anamount effective for reducing the density of the biscuits resulting frombaking the mixture.
 13. The method of claim 12 wherein the amount ofpuroindoline added is between 0.02 and 5% by weight relative to theweight of the flour.
 14. The method of claim 12 wherein the flour has apuroindoline content greater than 0.2% of the dry weight of the flourwith the additional ingredients.
 15. The method of claim 14 wherein thepuroindoline content of the flour is between 0.2 and 2% of the dryweight of the flour.